Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5β1 and αvβ3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvβ3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.

αβ integrin is localized in a unique punctate distribution at the cell-substratum interface along the leading front of single, front-rear-polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of β integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α integrin retains its punctate distribution. Moreover, in β integrin knockdown cells, we observe a loss of β integrin from focal adhesions and an enhanced association with α integrin. We confirmed the switch in the β integrin binding partner of α integrin in the knockdown cells by immunoprecipitation. We next investigated the role of β integrin in collective cell migration. Wounded monolayers of β integrin knockdown cells exhibit reduced collective migration compared with controls. When we forced expression of β integrin in the leader cells of wounded monolayers, collective migration was restored. Similarly, forced expression of β integrin in primary rat alveolar epithelial cells also promotes collective cell migration. In addition, we interrogated the pathway by which β integrin regulates A549 cell-directed migration. Constitutively active Ras-related C3 botulinum toxin substrate 1 rescues motility defects resulting from β integrin deficiency. Together, our results support the hypothesis that αβ integrin is a positive regulator of collective cell migration of A549 cells through influence on signal pathways in leader cells.

We find that clusters of β4 integrin, organized into distinct puncta, localize along vimentin filaments within lamellipodia at the cell edge of A549 cells, as assessed by interferometric photoactivated localization microscopy. Moreover, puncta and vimentin filaments exhibit a dynamic interplay in live cells, as viewed by structured-illumination microscopy, with β4 integrin puncta that associate with vimentin persisting for longer than those that do not. Interestingly, in A549 cells β4 integrin regulates vimentin cytoskeleton organization. When β4 integrin is knocked down there is a loss of vimentin filaments from lamellipodia. However, in these conditions, vimentin filaments instead concentrate around the nucleus. Although β4 integrin organization is unaffected in vimentin-deficient A549 cells, such cells move in a less-directed fashion and exhibit reduced Rac1 activity, mimicking the phenotype of β4 integrin-deficient A549 cells. Moreover, in vimentin-deficient cells, Rac1 fails to cluster at sites enriched in α6β4 integrin heterodimers. The aberrant motility of both β4 integrin and vimentin-deficient cells is rescued by expression of active Rac1, leading us to propose that complexes of β4 integrin and vimentin act as signaling hubs, regulating cell motility behavior.

During wound healing of the skin, keratinocytes disassemble hemidesmosomes and reorganize their actin cytoskeletons in order to exert traction forces on and move directionally over the dermis. Nonetheless, the transmembrane hemidesmosome component collagen XVII (ColXVII) is found in actin-rich lamella, situated behind the lamellipodium. A set of actin bundles, along which ColXVII colocalizes with actinin4, is present at each lamella. Knockdown of either ColXVII or actinin4 not only inhibits directed migration of keratinocytes but also relieves constraints on actin bundle retrograde movement at the site of lamella, such that actin bundle movement is enhanced more than 5-fold. Moreover, whereas control keratinocytes move in a stepwise fashion over a substrate by generating alternating traction forces, of up to 1.4 kPa, at each flank of the lamellipodium, ColXVII knockdown keratinocytes fail to do so. In summary, our data indicate that ColXVII-actinin4 complexes at the lamella of a moving keratinocyte regulate actin dynamics, thereby determining the direction of cell movement.-Hiroyasu, S., Colburn, Z. T., Jones, J. C. R. A hemidesmosomal protein regulates actin dynamics and traction forces in motile keratinocytes. FASEB J. 30, 2298FASEB J. 30, -2310FASEB J. 30, (2016. www.fasebj.org

The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton and matrix adhesions. In this study we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via an shRNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower than their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics and directed migration.